A bioreactor may refer to a device or system meant to seed cells onto scaffolds and grow cells or tissues, under specific biochemical and/or mechanical conditions, in the context of cell culture. Therefore tissue development is facilitated by bioreactors. In this context, bioreactors are devices engineered to deliver appropriate spatial and temporal nutrient transport that may also incorporate mechanical or other physical stimulation, in a well-defined and controlled environment. Bioreactors that provide well-defined and controllable environments are useful for fundamental studies to optimize tissue growth, and it can act as production units for large-scale tissue fabrication and bioprocesses. Bioreactor systems can provide reliable model systems for fundamental studies of cell biology and play a key role in improving the quality of engineered tissues.
Different bioreactors have been reported in the literature, including mixed flasks [Vunjak-Novakovic et al 1996], rotating vessels [Freed et al 1998; Freed et al, 2000], perfused cartridges [Carrier et al, 2002], and bioreactors with different mechanical stimulation [Altman et al, 2001; Altman et al, 2002].
Bichamber bioreactors and oscillating bioreactors have also been described for application in osteochondral (OC) TE [Wendt et al, 2003; Chang et al, 2004; Valonen et al, 2010].
The main limitation of the current bioreactors for OC TE is that the newly formed tissue(s) is not homogeneously distributed within the bilayered scaffolds [Chen et al, 2013]. Furthermore, there are no bioreactors adapted for bilayered scaffolds that support different culture medium for each layer of the bilayered constructs, allowing inducing rotatory stimulus, compression and vertical movement to avoid cell sedimentation and undesired OC tissue malformation, at the same time.
Several patents have been granted based in the use of bioreactors for different applications. The following examples should be taken into account by their relevance in the area of this invention:
US2006141623 (A1) patent of 23 Jun. 2006 describes systems, modules, bioreactor and methods for the automated culture, proliferation, differentiation, production and maintenance of tissue engineered products in a general way. However, this system description doesn't refer to double chamber (a chamber with two compartments), to rotating movements of each chambers independently nor to all system turning movement.
WO 2007/012071 of 25 Jan. 2007 refers to a bioreactor device, and a method and system for fabricating tissues and growing cells and tissues in the bioreactor device. The bioreactor device includes a bioreactor chamber for containing a sample, where sample growth in response to mechanical, electrical, and biofactor stimulation is monitored through one or more optical ports. Embedded sensors are provided for measuring fluid pressure, pH, temperature, and oxygen tension. The bioreactor device can receive different types of mechanical loadings, including fluid shear, hydrostatic pressure, matrix compression, and rotation. The system does not present a double chamber for bilayered structures.
WO Patent 2008/098165 of 14 Aug. 2008 refers to oscillating cell culture bioreactor. The bioreactor has a gas permeable, closed-loop chamber for cell or tissue culture, and an oscillating means for moving the gas permeable, closed-loop chamber bi-directionally along an axis horizontal to an axis normal to the closed-loop chamber to force convection of cells and fluid in the gas permeable, closed-loop chamber. The bioreactor optionally includes a tissue engineering scaffold, an inlet means, an outlet means, and integrated sensors. Another aspect provides a bioreactor having a plurality of gas permeable, closed-loop chambers for cell or tissue culture.
EP Patent 1990402 of 12 Nov. 2008 describes a bioreactor to apply mechanical forces as an anabolic stimulus. The invention describes a bioreactor designed to be used in the field of cytomechanics. The system comprises an electro-active polymer actuator, also known as artificial muscle, which is bonded to a deformable, water impermeable biocompatible substrate/matrix. This layered structure is the flexible membrane of the bioreactor.
U.S. Pat. No. 7,604,987 of 20 Oct. 2009 relates to a bioreactor presenting a chamber for containing cells or tissue cultures within a culture medium. The bioreactor also comprises a detector capable of detecting a change in one or more metabolites associated with growth of the cell or tissue cultures within the chamber and a chamber drive capable of rotating the chamber at a first speed about a first axis and a second speed about a second axis, the second axis being disposed at an angle relative to the first axis. In use, the magnitude of the first speed and the second speed are independently variable of each other. However, this bioreactor is composed just by one chamber for all structures samples, preventing co-culturing cells with two different media separately in each structure and each structure have no independent chamber container.
WO Patent 2010/040699 of 15 Apr. 2010 relates to multi-culture bioreactor system that can maintain stem cells and differentiated cell types in physically isolated environments but can allow biochemical communication between these cells. Despite the system allows the co-culture and biochemical interaction between several chambers, these different chambers are physically isolated, and consequently does not allow obtaining integrated bi- or multi-layered cultured scaffolds on individual optimized environments.
WO Patent 2010/064943 of 11 Jun. 2010 describes a bi-directional continuous perfusion bioreactor for tri-dimensional culture of mammal tissue substitutes. The bioreactor induces mechanical cellular stimuli by creating shear forces caused by the flow perfusion using different pressure gradients, controlled by two pumps, a vacuum and a peristaltic one, positioned within a circular flow system designed for the bioreactor and also through rotational engines positioned in specific locations. This bioreactor is able to grow tissues of large dimensions, through the control of the perfusion and flow gradient within the scaffolds, thereby obtaining both access of the nutrients to the interior and the removal of the metabolic products of the interior of the material.
EP Patent 2236597 A1 of 6 Oct. 2010 presents a high-throughput sensorized bioreactor for applying hydrodynamic pressure and shear stress stimuli on cell cultures. This hydrodynamic pressure is generated inside at least one culture chamber by means of variation of the mean distance, with a controlled speed, of a surface relative to the surface on which the culture is positioned and between which the culture medium is free to flow.
CN 101899393 (A) patent of 1 Dec. 2010 belongs to the field of bone tissue engineering and cell mechanics relating to a dynamic load and recirculating perfusion bioreactor, which comprises a main body, a sliding sleeve module and a three-dimensional culture cabin module. The invention comprises a piezoelectric ceramic that penetrates into the three-dimensional culture cabin through the hole. The moving end of the piezoelectric ceramic is connected with a loading rod and the culture cabin main body is also provided with a liquid inlet and a liquid outlet which are externally connected with a peristaltic pump. The bioreactor does not perform rotational movements nor is adapted for dual chamber.
EP Patent 2151491 A3 of 23 Nov. 2011 discloses a multichamber bioreactor, however this system has not rotational movement, which is a limitation regarding homogenization of culture, because cell sedimentation can occur by gravity.
CN102796664 patent of 28 Nov. 2011 is related with a human body ligament tissue engineering bioreactor that can perform tension/compression and two-way torsional multidimensional stress loading. Moreover, it can automatically detect the loosening condition of the bracket and perform bracket loosening on-line compensation; and has the characteristics of simple and compact structure and high use convenience. This bioreactor does not perform inversion of the culture chamber and is not applied to bilayered structures.
US2012035742 (A1) patent of 9 Feb. 2012 describes methods, devices and systems for bone tissue engineering using a bioreactor to culture human bone grafts. This bioreactor is composed by a unique chamber without rotating movements.
WO 2013103306 A1 patent of 11 Jul. 2013 refers to a bioreactor composed of watertight chamber and internal matrix for the generation of cellularized medical implants. The bioreactor's internal matrices and external chamber are simultaneously manufactured, preferably through a rapid prototyping process. The system is also characterized by an external chamber possessing a shape adapted to the shape of the implant to be cellularized (internal matrix) enabling a greater efficiency in the cellular colonization and culture of the implant. This bioreactor is not adapted to perform mechanical stimulus nor real time monitoring.
CN203269948 patent of 6 Nov. 2013 describes a device for tendon and tendon sheath in vivo co-culture construction. The device comprises a tissue engineering tendon and tendon sheath co-culture bioreactor, a control unit and a measuring unit, wherein the tissue engineering tendon and tendon sheath co-culture bioreactor comprises tissue engineering tendon and tendon sheath culture units and a pneumatic tendon sliding mechanism. The system comprises also monitoring sensors for pressure and flow. The system does not present different speed rotations for the several chambers.
US2014030762 (A1) of 30 Jan. 2014 relates to a bioreactor for cell culture on a three-dimensional structure, comprising one culture chamber, the inner walls of which form a vertical duct, with a diameter that widens regularly form the duct inlet to the duct outlet, means enabling the culture medium to flow in said vertical duct. The invention also relates to the advantageous use of these bioreactors in tissue engineering, for the production of tissue grafts, notably a bone or cartilage graft. This patent does not describe dual-chambers, rotational movements or bilayered structures culture.